Chen, Chih-Cheng; (1997) Molecular cloning and characterisation of a sensory neurone-specific ATP-gated channel (P2X3). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

ATP has been shown to directly activate sensory neurones in culture and evoke a sensation of pain on human blister bases. Molecular cloning of P2X3 showed that it is a sensory neurone-specific ATP-gated channel. The recombinant P2X3 thus provides a possible connection between ATP and nociception. The aim of this thesis is to characterise the expression pattern and function of the cloned P2X3 receptor. The full length of the P2X3 transcript is 3.8 kb, with a 397 amino acid coding region. The primary sequence of P2X3 shows a 36-48% homology to other P2X receptors and comprises a similar structure of two transmembrane domains, short intracellular N- and C-terminals, a large extracellular loop, and ten conserved cysteine residues. Northern analysis and in situ hybridisation showed that the P2X3 transcript is exclusively expressed in a subset of sensory neurones which are mainly peripherin positive and capsaicin sensitive small diameter neurones. Such neurones contain thin or nonmyelinated axons which are nociceptive fibres. The electrophysiological properties of the P2X3 receptor was examined using an oocyte expression system. By using a two electrode voltage clamp, an ATP evoked inward current with fast desensitisation can be detected in oocytes injected with P2X3 complementary RNA. In oocytes, the mechanism of P2X3 desensitisation was found to be calcium-dependent and regulated by calcineurin. Developmental studies showed that the expression of P2X3 transcript is as early as the period of sensory neurogenesis at rat E11.5 embryos. For further investigation of P2X3 expression and function, monoclonal antibodies against the P2X3 receptor were generated and characterised. In addition, a DRG-specific proton-gated channel ASIC β was also cloned. It belongs to the DEG/ENaC family whose structure is similar to P2X3 with two transmembrane spanning but with a larger extracellular loop and different conserved cysteine residues. Low pH (pH < 6.5) was found to evoke a non-selective inward cation flux in ASIC β transfected COS cells. As both ATP and protons are important chemical mediators in inflamed tissues, the corresponding ligand-gated channels P2X3 and ASIC β may be good targets for analgesic drug development.

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